Three-component protein complexes spanning two membranes are universally spread among Gram-negative bacteria and have been implicated in such diverse range of transport functions as delivery of virulence factors into the hosts, secretion of signaling molecules and protection of bacterial cells against structurally diverse antimicrobial agents. A remarkable feature of these transporters is that the substrate transfer occurs across two membranes directly into external medium bypassing the periplasm. However the biochemical mechanism of such transport remains unclear. The major bottleneck in characterization of three-component transporters is that traditional biochemical methods cannot be readily applied to two-membrane systems. Our long-term goal is to understand how three-component transporters function in the context of two membranes. The objective of this application is to develop a novel experimental approach to study the biochemical mechanism of three-component transporters from Gram-negative pathogens. Our central hypothesis is that elimination of topological and heterogeneity problems of reconstituted proteoliposomes will significantly advance studies of two-membrane transporters by various biochemical techniques. The approach used to test this hypothesis is to reconstitute the macrolide and enterotoxin transporter MacAB-TolC into high-density lipoprotein particles and to develop real-time assays to study the mechanism of this transporter. We will pursue two specific aims: (i) Reconstitute MacA, MacB and TolC into lipid nanodiscs and characterize their biochemical activities;(ii) Develop real-time binding assays to study assembly of MacAB-TolC complex. The expected outcome of the proposed studies is development of quantitative assays to study multi-protein transporters with components located in two different membranes and characterization of assembly of such transporters in real-time. This contribution is significant because two-membrane transporters are responsible for the intrinsic antibiotic resistance and virulence of Gram-negative pathogens and their assembly and functions are targeted in development of effective antimicrobial agents. PUBLIC HEALTH RELEVANCE: Gram-negative pathogens cause devastating infections in humans and are intrinsically resistant to a broad range of clinically important antibiotics. This proposal is focused on the development of experimental tools to study the molecular mechanisms of proteins that contribute in the major way to both bacterial virulence and antibiotic resistance.